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Found: 26 174
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A Nucleic Acid/Gold Nanorod-Based Nanoplatform for Targeted Gene Editing and Combined Tumor Therapy.
The CRISPR/Cas9 gene-editing system has become a promising strategy for tumor therapy with its powerful oncogene-editing ability. However, the efficient delivery of sgRNA/Cas9 complex into target tumor cells remains a challenge. Herein, we report a facile strategy for the construction of an sgRNA/Cas9 complex co-assembled nanoplatform for targeted gene editing and combined tumor therapy. In our design, the TAT peptide and thiolated DNA linker functionalized gold nanorod can efficiently load the sgRNA/Cas9 complex through the hybridization between the 3' overhang of sgRNA and the DNA linker. Due to the integration of an active cell targeting group (aptamer) and nuclear targeting peptide (TAT), the multifunctional nanoplatform can elicit the targeted cellular internalization and efficient nuclear targeting transportation to realize endogenous RNase H activated gene editing of the tumor-associated gene polo-like kinase 1 (PLK1). With mild photothermal treatment, this sgRNA/Cas9 complex loaded nanoplatform achieved efficient inhibition of tumor cell proliferation. This multifunctional nanocarrier provides a new strategy for the development of combined tumor therapy.
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Engineering salt-tolerant Cas12f1 variants for gene-editing applications.
CRISPR has revolutionized the field of genome editing in life sciences by serving as a versatile and state-of-the-art tool. Cas12f1 is a small nuclease of the bacterial immunity CRISPR system with an ideal size for cellular delivery, in contrast to CRISPR-associated (Cas) proteins like Cas9 or Cas12. However, Cas12f1 works best at low salt concentrations. In this study, we find that the plasticity of certain Cas12f1 regions (K196-Y202 and I452-L515) is negatively affected by increased salt concentrations. On this line, key protein domains (REC1, WED, Nuc, lid) that are involved in the DNA-target recognition and the activation of the catalytic RuvC domain are in turn also affected. We suggest that salt concentration should be taken in to consideration for activity assessments of Cas engineered variants, especially if the mutations are on the protospacer adjacent motif interacting domain. The results can be exploited for the engineering of Cas variants and the assessment of their activity at varying salt concentrations. We propose that the K198Q mutation can restore at great degree the compromised plasticity and could potentially lead to salt-tolerant Cas12f1 variants. The methodology can be also employed for the study of biomolecules in terms of their salinity tolerance.Communicated by Ramaswamy H. Sarma.
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Cationic lipid nanoparticle-mediated delivery of a Cas9/crRNA ribonucleoprotein complex for transgene-free editing of the citrus plant genome.
A lipofectamine-mediated transfection protocol for DNA-free genome editing of citrus protoplast cells using a Cas9/gRNA ribonucleoprotein (RNP) complex resulted in the production of transgene free genome edited citrus.
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Genome editing technologies drive the development of pig genetic improvement.
Nuclease-mediated genome editing technologies contribute to the rapid advances in life sciences via the ability to edit the genomes within living cells, and present a new era for porcine genetic improvement. In this review, we introduce the development of various genomic editing technologies, particularly CRISPR/Cas9 strategies and characteristics of various naturally occurring and artificially engineered CRISPR enzymes. Also, we summarize progress in pig genetic improvement mediated by genome editing, especially those associated with meat quality traits and anti-virus resistance. We highlight the challenges in the implementation of pig genetic improvement and the prospects of pig genetic breeding based on genome editing technologies.
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Restored Collagen VI Microfilaments Network in the Extracellular Matrix of CRISPR-Edited Ullrich Congenital Muscular Dystrophy Fibroblasts.
Collagen VI is an essential component of the extracellular matrix (ECM) composed by α1, α2 and α3 chains and encoded by COL6A1, COL6A2 and COL6A3 genes. Dominant negative pathogenic variants in COL6A genes result in defects in collagen VI protein and are implicated in the pathogenesis of muscular diseases, including Ullrich congenital muscular dystrophy (UCMD). Here, we designed a CRISPR genome editing strategy to tackle a dominant heterozygous deletion c.824_838del in exon 9 of the COL6A1 gene, causing a lack of secreted collagen VI in a patient's dermal fibroblasts. The evaluation of efficiency and specificity of gene editing in treating patient's fibroblasts revealed the 32% efficiency of editing the mutated allele but negligible editing of the wild-type allele. CRISPR-treated UCMD skin fibroblasts rescued the secretion of collagen VI in the ECM, which restored the ultrastructure of the collagen VI microfibril network. By using normal melanocytes as surrogates of muscle cells, we found that collagen VI secreted by the corrected patient's skin fibroblasts recovered the anchorage to the cell surface, pointing to a functional improvement of the protein properties. These results support the application of the CRISPR editing approach to knock out COL6A1 mutated alleles and rescue the UCMD phenotype in patient-derived fibroblasts.
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CRISPR-Cas9 genome editing in Corallochytrium limacisporum,a key species for understanding animal origins.
Microbial holozoans are the closest unicellular relatives of animals. They share a substantial gene repertoire with animals and exhibit complex life cycles. Studying these organisms is crucial for understanding the evolution of multicellularity, and significant progress has been made in uncovering key aspects of the biology of the four microbial holozoans lineages: choanoflagellates, filastereans, ichthyosporeans and corallochytreans. However, reverse genetic tools are still lacking in corallochytreans, one of the earliest-branching holozoan lineages and the only known group with both coenocytic and binary fission development. Here, we present CRISPR-Cas9-mediated gene inactivation and point mutation methodologies in the corallochytrean Corallochytrium limacisporum. As a proof of concept, we inactivated the fkb12 gene, a component of the mTOR pathway, conferring rapamycin resistance, and introduced a point mutation in sdhB, encoding a subunit of succinate dehydrogenase, conferring carboxin resistance. Our results demonstrate the presence of both non-homologous end-joining and homology-directed repair pathways in C. limacisporum and shows an editing efficiency of approximately 2%. Furthermore, simultaneous gene targeting revealed a co-editing frequency of approximately 20%. Finally, this study establishes unequivocally that C. limacisporum is haploid, making it an ideal model for genetic studies and gene editing applications to unravel the molecular mechanisms involved in animal origins.
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"Primed for Repair: Harnessing Hypoxia, Mechanobiology, and Gene Editing to Enhance MSC Potency and Clinical Translation".
Mesenchymal Stem/Stromal Cells (MSCs) hold substantial promise for regenerative medicine; however, their clinical translation has been limited by inconsistent efficacy, poor survival after transplantation, and highly variable functional potency. Preconditioning, defined as the ex vivo manipulation of MSCs before administration, has emerged as a key strategy to address these shortcomings. This review catalogues and critically evaluates the major preconditioning techniques developed between 2000 and 2025, aimed at improving the overall fitness of undifferentiated MSCs or steering their commitment toward specific therapeutic lineages, including osteogenic, chondrogenic, adipogenic, cardiomyogenic, and neurogenic outcomes. We grouped these strategies into two broad categories: (1) non-genomic manipulations, which rely on transient exposure to physical, chemical, or biological cues, such as hypoxia, mechanical forces, small molecules, cytokine treatment, and extracellular matrix engineering; and (2) genomic or gene-modifying manipulations, which involve more stable alterations through microRNA/siRNA modulation, CRISPR/Cas-based gene editing, and viral or non-viral gene transfer. For each modality, we synthesized the relevant molecular mechanisms, evaluated evidence from in vitro, preclinical, and clinical studies, and examined key translational barriers. We also considered important evaluation metrics, including safety, engraftment efficiency, and functional recovery, across disease models. Finally, we integrate perspectives on the evolving clinical trial landscape, regulatory developments from the FDA and EMA, and the intellectual property environment, concluding with a discussion of future research priorities that may accelerate the development of next-generation, potency-enhanced MSC therapeutics.
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Generation of mutant pigs by lipofection-mediated genome editing in embryos.
The specificity and efficiency of CRISPR/Cas9 gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos. Given the limited time window for delivery, faster and more reliable methods to introduce Cas9-gRNA ribonucleoprotein complexes (RNPs) into target embryos are needed. In pigs, somatic cell nuclear transfer using gene-modified somatic cells and the direct introduction of gene editors into the cytoplasm of zygotes/embryos by microinjection or electroporation have been used to generate gene-edited embryos; however, these strategies require expensive equipment and sophisticated techniques. In this study, we developed a novel lipofection-mediated RNP transfection technique that does not require specialized equipment for the generation of gene-edited pigs and produced no detectable off-target events. In particular, we determined the concentration of lipofection reagent for efficient RNP delivery into embryos and successfully generated MSTN gene-edited pigs (with mutations in 7 of 9 piglets) after blastocyst transfer to a recipient gilt. This newly established lipofection-based technique is still in its early stages and requires improvements, particularly in terms of editing efficiency. Nonetheless, this practical method for rapid and large-scale lipofection-mediated gene editing in pigs has important agricultural and biomedical applications.
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High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells.
UNLABELLED: Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA, that defines the genomic target, need to be present and co-localized within the nucleus for efficient gene-editing to occur. This work describes a new high-throughput screening platform for the optimization of CRISPR-Cas9 delivery strategies. By exploiting high content image analysis and microcontact printed plates, multi-parametric gene-editing outcome data from hundreds to thousands of isolated cell populations can be screened simultaneously. Employing this platform, we systematically screened four commercially available cationic lipid transfection materials with a range of RNAs encoding the CRISPR-Cas9 system. Analysis of Cas9 expression and editing of a fluorescent mCherry reporter transgene within human embryonic kidney cells was monitored over several days after transfection. Design of experiments analysis enabled rigorous evaluation of delivery materials and RNA concentration conditions. The results of this analysis indicated that the concentration and identity of transfection material have significantly greater effect on gene-editing than ratio or total amount of RNA. Cell subpopulation analysis on microcontact printed plates, further revealed that low cell number and high Cas9 expression, 24h after CRISPR-Cas9 delivery, were strong predictors of gene-editing outcomes. These results suggest design principles for the development of materials and transfection strategies with lipid-based materials. This platform could be applied to rapidly optimize materials for gene-editing in a variety of cell/tissue types in order to advance genomic medicine, regenerative biology and drug discovery. STATEMENT OF SIGNIFICANCE: CRISPR-Cas9 is a new gene-editing technology for "genome surgery" that is anticipated to treat genetic diseases. This technology uses multiple components of the Cas9 system to cut out disease-causing mutations in the human genome and precisely suture in therapeutic sequences. Biomaterials based delivery strategies could help transition these technologies to the clinic. The design space for materials based delivery strategies is vast and optimization is essential to ensuring the safety and efficacy of these treatments. Therefore, new methods are required to rapidly and systematically screen gene-editing efficacy in human cells. This work utilizes an innovative platform to generate and screen many formulations of synthetic biomaterials and components of the CRISPR-Cas9 system in parallel. On this platform, we watch genome surgery in action using high content image analysis. These capabilities enabled us to identify formulation parameters for Cas9-material complexes that can optimize gene-editing in a specific human cell type.
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Recent Advances in CRISPR/Cas9-Mediated Genome Editing in Dictyostelium.
In the last 30 years, knockout of target genes via homologous recombination has been widely performed to clarify the physiological functions of proteins in Dictyostelium. As of late, CRISPR/Cas9-mediated genome editing has become a versatile tool in various organisms, including Dictyostelium, enabling rapid high-fidelity modification of endogenous genes. Here we reviewed recent progress in genome editing in Dictyostelium and summarised useful CRISPR vectors that express sgRNA and Cas9, including several microorganisms. Using these vectors, precise genome modifications can be achieved within 2⁻3 weeks, beginning with the design of the target sequence. Finally, we discussed future perspectives on the use of CRISPR/Cas9-mediated genome editing in Dictyostelium.
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